The ameliorative role of ascorbic acid against blood disorder, immunosuppression, and oxidative damage of oxytetracycline in rainbow trout (Oncorhynchus mykiss).

This experiment was aimed to determine the possible beneficial effects of dietary ascorbic acid (AA) on hematological indices, immune responses, and antioxidative capacity of Oncorhynchus mykiss treated with antibiotic oxytetracycline (OTC). A total of 150 fish were divided evenly among five experimental groups (30 fish of each, in 3 replicates) receiving diets containing OTC (0 and 100 mg per kg fish weight) and AA (100, 200, 400, and 800 mg per kg fish diet) for 28 days. Treatments include group A or control (100 mg AA without OTC), group B (100 mg AA with OTC), group C (200 mg AA with OTC), group D (400 mg AA with OTC), and group E (800 mg AA with OTC). The results obtained showed that the hematological indices (red blood cells, white blood cells, hematocrit, hemoglobin, and neutrophils), immunological parameters (plasma lysozyme, plasma complement, and skin mucus alkaline phosphatase activities), and antioxidant enzymes activities (superoxide dismutase and catalase) were significantly decreased by OTC in O. mykiss fed control diet (P < 0.05). The results also revealed that OTC significantly increased the activity of biochemical enzymes (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) in the plasma of O. mykiss fed control diet (P < 0.05). However, in comparison to the control diet, feeding fish with higher amounts of AA (400 and 800 mg/kg diet) significantly restored the hematological, immunological, and antioxidative responses in OTC-treated groups (p < 0.05). These findings show that the dietary supplementation of AA at 400 or 800 mg/kg diet is beneficial in relieving O. mykiss from OTC-induced oxidative stress and immunosuppression.

Foodborne fluoxetine impacts the immune response in rainbow trout (Oncorhynchus mykkis).

The aim of this study was to reveal the effects of foodborne fluoxetine on morphological and condition profile, hematological profile, biochemical and oxidative stress indices on juvenile rainbow trout. The study was performed according to OECD Guideline No. 215. Fluoxetine was incorporated into Biomar 921 pellets at a dose of 0.047 mg/kg (environmental concentration), 0.577 mg/kg and 6.7 mg/kg. There was statistically significant change in hematological profile, including an increasing trend in neutrophil/lymphocyte ratio and a decreasing trend in the number of lymphocytes. Measurements of oxidative stress indicated decreased activity of the detoxifying enzyme glutathione-S-transferase in the liver and kidney. However, the measurement of GR, GPx, CAT, SOD activity, and TBARS showed no changes. Histopathological examination revealed damage to the proximal tubules of caudal kidney in exposed groups. This study confirms that fluoxetine has a significant effect on immune response.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout.

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Modulatory effect of dietary copper nanoparticles and vitamin C supplementations on growth performance, hematological and immune parameters, oxidative status, histology, and disease resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).

Copper and vitamin C are micronutrients needed for the living organism's functions. Vitamin C has a great effect on the immune system of fish. The present study aimed to evaluate the effects of dietary copper nanoparticles (Cu-NPs) and vitamin C (VC) supplementations on rainbow trout (Oncorhynchus mykiss) juveniles. So, 216 rainbow trout juveniles were randomly assigned to six groups with trial diets supplemented with Cu-NPs and VC including 0/0 (T1, control diet), 0/250 (T2), 0/500 (T3), 2/250 (T4), 2/500 (T5), and 2/0 (T6) mg Cu-NPs/VC per kg diet. After the feeding trial for 60 days, the fish were challenged with Yersinia ruckeri, and the survival rate was calculated for 15 days. Based on the data analysis, weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), lysozyme, alternative complement activity (ACH50), hematocrit (Hct), hemoglobin (Hb), and mean corpuscular volume (MCV) were significantly (p < 0.05) increased in the fish fed on T4 and T5 diets compared with the control group. Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were significantly (p < 0.05) decreased in the fish fed with diets contain Cu-NPs and VC (T4 and T5). The expressions of TNF-α, IL-1ß, IL-10, SOD, CAT, and GPX genes were significantly (p < 0.05) decreased in the fish fed on T3, T4, and T5 diets versus the control. In addition, the dietary Cu-NPs and VC supplementations significantly enhanced resistance against pathogens and led to the control of infection in rainbow trout. In conclusion, Cu-NPs and VC administered as feed additives at 2/250-500 mg/kg elevated the growth performance, antioxidant capacity, and health of rainbow trout.

Mooseer (Allium hirtifolium) boosts growth, general health status, and resistance of rainbow trout (Oncorhynchus mykiss) against Streptococcus iniae infection.

In large-scale aquaculture, the fast growth rate of fish is positively influenced by feed additives such as medicinal plants. This is however; infectious disease may reduce fish growth and cause devastating economic loss. The present study investigated in vitro antibacterial efficacy of Mooseer (Allium hirtifolium) extract against Streptococcus iniae and its in vivo effects on growth, biochemical parameters, innate immunity of rainbow trout (Oncorhynchus mykiss). Therefore, six experimental diets were designed to include different levels of Mooseer from zero (as control), 5, 10, 15, 20, and 25 g per kg diet respectively referred to as M1 to M5. Results from the antibacterial evaluation showed that Mooseer extract inhibits S. iniae growth with MIC and MBC values of 128 and 256 µg ml-1. Appreciable results were obtained in the groups supplemented with Mooseer. Mooseer enhanced growth performance, and modulated serum biochemical and immunological parameters (total protein, albumin, triglyceride, glucose, cortisol, cholesterol, lysozyme, Ig, ACH50, ALP, and protease activity), and liver enzymes (ALT, AST and ALP). The greatest effects were found for higher doses of Mooseer supplementation (M4 and M5). Meanwhile, results from the survival rate of fish challenged with S. iniae showed higher survival in M2 and M4 treatments. The present findings suggest the beneficial use of Mooseer in rainbow trout diet, with 20 g kg-1 inclusion as the recommended dose.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4-5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/ß or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family Salmonidae) include two key teleost species for aquaculture, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in Salmo, the majority with a close counterpart in Oncorhynchus. Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

RNA form baker's yeast cultured with and without lipopolysaccharide (LPS) modulates gene transcription in an intestinal epithelial cell model, RTgutGC from rainbow trout (Oncorhynchus mykiss).

The objective of this study was to evaluate if the intestinal RTgutGC cell line could be suitable for research on dietary ingredients and their function as modulators of inflammation during lipopolysaccharide (LPS) induced stress. The RTgutGC cells cultured together with RNA from baker's yeast, reached confluency after 72 h. The cells were grown in either compete L-15 (CM) or nutrient deprived L-15 (DM). Then, the RTgutGC cells were exposed to LPS or RNA from baker's yeast, either alone, or in combination, in CM or DM. All cultures were harvested following LPS challenge for 48 h and 72 h. LPS induced transcription of Interleukin 1ß (IL-1ß), Interleukin -8 (IL-8), Toll like receptor 3 (TLR3), interferon regulating factor 3 (irf3), Nuclear factor Ä¸ß (NFĸß), one of the multidrug transporters, ABCC2, and glutamine synthase 1 (GLS01) in RTgutGC cells at one or both sampling points (48 h and/or 72 h post LPS challenge). RNA from baker's yeast in culture alone, (cultured 120 h and 144 h with RTgutGC cells and harvested at the respective LPS sampling points) induced transcription of INF1, TNFα and ticam/trif, not induced by LPS. In addition, RNA from baker's yeast affected IL-1ß, TLR3, irf3 and NFĸß, comparable to the responses triggered by LPS. RNA from baker's yeast alone did not affect ABCC2 or GLS01 transcriptions in this set up. So, LPS and RNA from baker's yeast affects distinct but also common gene transcripts in this intestinal cell line. Culturing RTgutGC cells in DM, adding a combination of LPS and RNA from baker's yeast, reduced IL-1ß transcription compared to cells grown in CM, 48 h and 72 h post LPS challenge. Also, in RTgutGC cells, grown in DM, the LPS induced transcription of ABCC2 declined, measured 48 h post LPS challenge. Possibly indicating that optimal transcription of IL-1ß and ABBC2 in RTgutGC cells, cultured over time, requires access of adequate nutrients under stressful condition. RNA from baker's yeast induced INF1 transcription in the RTgutGC cells, regardless if the medium was complete or deprived of nutrients. However, culturing RTgutGC cells in DM enriched with RNA from baker's yeast for a longer period of time (120 h, 144 h), seemed beneficial for INF1 transcription.

Dietary supplementation with dandelion (Taraxacum officinale) flower extract provides immunostimulation and resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchusmykiss).

Dandelion flower is a well-known phytomedicine due to its therapeutic effects on dyspepsia, bile duct disorders, spleen and liver complaints, and inflammatory diseases. This study aimed to assess the effect of dietary dandelion flower extract (DFE) on skin mucosal and humoral immunity, immune-related gene expression, and disease resistance in rainbow trout. The fingerlings (N = 300, 20.1 ± 0.9 g) were randomly assigned to 15 tanks and fed with different levels of DEF including 0 (control), 1, 2, 3, and 4 g/kg for 56 days. At the end of the trial, the total leukocyte and lymphocyte counts were significantly enhanced in DFE-added groups compared to the control group. Complement components, immunoglobulin M, total protein, and lysozyme showed significantly higher values in the fish treated with dietary DFE at 3 g/kg than others (P < 0.05). The enzymatic activities of the skin mucus samples in the fish fed dietary levels of DFE were significantly higher than the control fish. Skin mucus protein was also improved significantly in 2 and 3 g/kg DFE compared to others. The transcription levels of interleukin-1ß and interleukin-6 genes were up-regulated in the fish fed with 3 and 4 g/kg DFE. Interleukin-8 and lysozyme gene expression levels were elevated in 3 g/kg DFE group compared to the control group. Fish mortality after challenging with Streptococcus iniae was significantly reduced from 43.33% in the control group to 73.33% in 3 g/kg DFE group. This study confirmed the beneficial effects of DFE as an immunostimulant in rainbow trout diet and the recommended dose of DFE is 2.49-2.74 g/kg based on the polynomial regression models.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout.

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Dietary effects of a low-molecular weight fraction (<10 kDa) from shrimp waste hydrolysate on growth performance and immunity of rainbow trout (Oncorhynchus mykiss): Employing nanodelivery systems.

Aquaculture by-products have been of great interest for producing protein hydrolysates with multiple biological activities. The present experiment was carried out to evaluate dietary effects of a low-molecular fraction (<10 kDa) from shrimp waste hydrolysate in forms of unprotected and nanocapsulated on growth and immunity of rainbow trout. Therefore, six diets were designed including a control diet (no supplementation), D1 (1 g kg-1 of unprotected fraction), D2 (1 g kg-1 chitosan nanocapsules), D3 (1 g kg-1 liposome nanocapsules), D4 (1 g kg-1 of fraction-loaded chitosan nanocapsules), D5 (1 g kg-1 of fraction-loaded liposome nanocapsules). Fish (0.91 ± 0.15 g) were fed with experimental diets until apparent satiation for six weeks followed by a 5-day experimental challenge with Streptococcus iniae. Results revealed that growth is strongly affected in fish receiving the fraction with D4 treatment showing the highest weight gain, SGR, final weight and the lowest FCR (p < 0.05). Nanocapsules without fraction did not show remarkable effects when compared to control group. In terms of serum and mucus immune parameters of lysozyme, complement activity, myeloperoxidase activity, and total protease, fish from D4 group showed the highest measured values followed by D5 (p < 0.05). Key immune related genes of IL-6 and TNF-α were noticeably up-regulated in fish from D1, D4, and D5 groups, which were consistent with survival rate after 5 days challenge with Streptococcus iniae. All together, the present findings highlighted the application of chitosan and liposome nanocarriers in aquaculture and potential of low-molecular weight fraction (<10 kDa) from shrimp wastes hydrolysate to improve growth performance and immune status of rainbow trout.

Oral administration of Sargassum polycystum extracts stimulates immune response and increases survival against Aeromonas hydrophila infection in Oncorhynchus mykiss.

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 µl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1ß gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.

Effects of hypoxia and hyperoxia on growth parameters and transcription levels of growth, immune system and stress related genes in rainbow trout.

Hypoxia and hyperoxia are disparate stressors which can have destructive influences on fish growth and physiology. It is yet to be determined if hypoxia and hyperoxia have a cumulative effect in aquatic ecosystems that affect biological parameters in fish, and to understand if this is associated with gene expression. Here we address whether growth performance and expressions of growth, immune system and stress related genes were affected by hypoxia and hyperoxia in fish. Rainbow trout was chosen as the study organism due to its excellent service as biomonitor. After an acclimatization period, fish were exposed to hypoxia (4.0 ± 0.5 ppm O2), normoxia (7.5 ± 0.5 ppm O2) and hyperoxia (12 ± 1.2 ppm O2) for 28 days. At 6 h, 12 h, 24 h, 48 h, 72 h and 28 days, samples were collected. Hypoxia and hyperoxia negatively affected weight gain (WG), specific growth rate (SGR), survival rate (SR) and feed conversion ratio (FCR). The best WG, SGR, SR and FCR values occurred in fish exposed to normoxia, whereas hypoxia was most suppressive on growth and hyperoxia showed intermediate suppression of these parameters. Gene expression analyses were performed in liver and results revealed that long term exposure caused reduced growth hormone-I (GH-I) and insulin like growth factor I-II (IGF I-II) levels in both hypoxia and hyperoxia-treated fish. Heat shock protein (HSP70) levels increased in both hypoxia and hyperoxia treatment, and both exposures caused elevation of leptin (LEP) expression in long-term exposure. Overall data indicate that both hypoxia and hyperoxia cause stress in rainbow trout and negatively affects growth parameters.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss.

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Evidence of Transcriptional Shutoff by Pathogenic Viral Haemorrhagic Septicaemia Virus in Rainbow Trout.

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-ß signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout.

Cell-mediated immune response against mycolic acids of Mycobacteroides salmoniphilum in rainbow trout Oncorhynchus mykiss.

Mycobacteriosis caused by Mycobacterium spp. causes economic damages to the world aquaculture industry. In mammals, mycolic acids contained in the cell wall of Mycobacterium spp. are presented by CD1b molecule as lipid antigens and induce cell-mediated immunity (CMI). Here, we investigated CMI responses against the mycolic acids of Mycobacterioides salmoniphilum in a CD1-lacking teleost fish, rainbow trout. After stimulation of trout leukocytes with mycolic acids, the number and percentage of CD8α+ T cells increased. Fish immunized with mycolic acids showed an up-regulation of IFN-γ. Further, in vitro re-stimulation of leukocytes derived from immunized fish resulted in proliferation of CD8α+ cells. These data suggest that mycolic acids are recognized as lipid antigens resulting in an activation of rainbow trout CD8α+ cells and up-regulation of the Th1 cytokine IFN-γ. The mycolic acids are promising candidates for vaccines to activate CD8α+ T cells against fish mycobacteriosis.

Oral Administration of Lactococcus lactis Producing Interferon Type II, Enhances the Immune Response Against Bacterial Pathogens in Rainbow Trout.

Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.